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Name the stages of development in the erythrocytic series, using two systems of nomenclature.

Rubriblast, Prorubricyte, Rubricyte, Metarubricyte, Reticulocyte, and Erythrocyte.

Pronormoblast, Basophilic normoblast, Polychromatic normoblast, Orthrochromic normoblast, Polychromatophilic erythrocyte, and Erthyrocyte.


What is the last nucleated stage in development of the erythrocyte?

The metarubricyte/orthrochromic normoblast. The nucleus is "pitted" by the spleen.


What is asynchronous erythropoiesis?

Erythropoiesis in which the nucleus and cytoplasm mature at different rates. For example, in iron deficiency anemia the cytoplasm appears younger than the nucleus because of insufficient iron for hemoglobin synthesis and the mature erythrocytes are microcytic and hypochromic. In megaloblastic erythropoiesis the nucleus lags behind the cytoplasm in development because of a deficiency of vitamin B12 or folic acid and the mature erythrocytes are macrocytic.


Describe the normal mature erythrocyte.

A buff-colored biconcave disk, 6-8 microns in diameter, with an area of central pallor approximately 1/3 the diameter of the cell. The normal RBC is described as normocytic, normochromic. Cells greater than 9 microns in diameter are called macrocytes and those less than 6 microns are called microcytes. Cells whose central pallor is greater than 1/3 the diameter of the cell are described as hypochromic.


What is the best way to judge the size of an erythrocyte on a Wright-stained smear?

A normocytic RBC is approximately the same size, or slightly smaller, than the nucleus of a mature lymphocyte (8-9 microns). It is important to be in the correct area of the smear when evaluating RBC morphology--an area where the RBCs are just barely touching (approximately 200 RBCs in a 1,000x field). RBCs in the feathered edge appear macrocytic and lack central pallor. In the thick part of the smear RBCs appear microcytic and may seem to form rouleaux.


What is the first sign of accelerated erythropoiesis?

An increased reticulocyte count.


Define anisocytosis.

Variation in the size of the RBCs. This is a non-specific finding in anemia. If the RDW (red cell distribution width) is increased, anisocytosis should be seen on the blood smear.


Define poikilocytosis.

Variation in the shape of the RBCs. Some shapes are related to specific diseases such as sickle cells; others are nonspecific findings.


What are schistocytes?

They are RBC fragments that are seen with disseminated intravascular coagulation (DIC), hemolysis, burns, microangiopathic hemolytic anemia, and in patients with artificial heart valves.


What is a drepanocyte?

A sickle cell. These cells are seen in sickle cell anemia but not usually in sickle cell trait.


What is the significance of hemoglobin C crystals?

These hexagonal, dark-staining crystals with blunt ends ("Washington monuments") are seen in some patients with hemoglobin C disease (CC). Frequently the cell membrane cannot be seen and the crystal appears to be free. Hemoglobin C crystals are not seen with hemoglobin C trait (AC),


What are dacryocytes?

Tear-drop shaped RBCs. They are seen in myelofibrosis and a variety of anemias.


How can crenated RBCs be differentiated from burr cells?

Burr cells may have more pointed projections as compared to the knobby projections of crenated cells, but the difference may be minimal. If the abnomality is seen in nearly all cells in the thin part of the smear, it is most likely artifact (crenation) due to slow drying of the smear or over-anticoagulation. Burr cells are seen in variable numbers throughtout the smear.


What is the clinical significance of burr cells?

They are seen with liver disease, uremia, hemolytic anemias, TTP, DIC, carcinoma of the stomach, and pyruvate kinase deficiency.


What is hypochromia?

A condition in which the area of central pallor is greater than 1/3 the diameter of the RBC.


What is a codocyte?

A target cell. This cell resembles a bull's-eye, with a peripheral rim of hemoglobin and a dark central hemoglobin-containing area. Target cells are thin cells that appear as "Mexican hats" with a scanning electron microscope. They are a non-specific finding and occur in a variety of anemias, especially the hemoglobinopathies such as sickle cell anemia and hemoglobin C disease.


What is a leptocyte?

A variation of a target cell in which the central portion is not completely detached from the other membrane. Leptocytes are associated with hepatic disorders, iron deficiency anemia, and thalassemia.


What are spherocytes?

RBCs that are spherical rather than biconcave. They have a diameter of 6-7 microns, stain darkly, and have no central pallor. They are the result of damage to the RBC membrane and have a shortened life-span. They are seen with hereditary spherocytosis, hemolytic anemia, ABO hemolytic disease of the newborn (but not with Rh hemolytic disease of the newborn), burns, and following the transfusion of stored blood. A few spherocytes may also be seen in normal blood as part of the aging process. Spherocytes may be erroneously reported if one examines the feathered edge of the blood smear since RBCs in that area lack central pallor. In hereditary spherocytosis, the MCV may be normal to low and the MCHC slightly increased.


On a blood smear spherocytes appear small in comparison to normal RBCs. Explain how their MCV may be normal.

The appearance of the cells on the blood smear is a 2-dimensional representation. Since their diameter is decreased, spherocytes appear small. MCV is a 3-dimensional measurement of cell volume. Although spherocytes are small in diameter, their volume is normal because they are spherical rather than biconcave.


What are stomatocytes?

RBCs with slit-like central pallor. They may be found in liver disease, alcoholism, electrolyte imbalance, and hereditary stomatocytosis. They are often just an artifact.


What is the clinical significance of marked polychromasia?

It is a sign of accelerated erythropoiesis. If a new methylene blue stain is performed on the blood, the reticulocyte count will be elevated.


What is basophilic stippling?

Multiple irregularly-shaped purple inclusions throughout the cytoplasm of the RBC. IT may be fine or coarse. Basophilic stippling is due to aggregates of RNA and is associated with abnormal heme synthesis (fine) and lead poisoning (coarse).


Which RBC inclusion is associated with lead poisoning?

Coarse basophilic stippling.


What are Heinz bodies?

Round inclusions, 1-3 microns in diameter, attached to the RBC membrane. They stain with supravital stains but not with Wright stain. They are precipitated denatured hemoglobin and are seen with G-6-PD deficiency and drug-induced hemolytic anemia.


What are Howell-Jolly bodies?

Round nonrefractile purple inclusions, 1-2 microns in diameter, that are seen in RBCs post-splenectomy and in a variety of anemias. There is usually only one per cell. They are nuclear remnants (DNA).


What are siderotic granules?

Small irregular dark-staining iron granules near the peripheral of the RBC, seen in a Prussian blue stain. They are celled Pappenheimer bodies when seen on a Wright stain.


Which of the following can be seen on a Wright stained blood smear: Heinz bodies, Howell Jolly bodies, reticulocytes, siderocytes?

Only Howell-Jolly bodies. Heinz bodies and reticulocytes are seen with supravital stains--Heinz bodies with crystal violet and reticulocytes with new methylene blue or brilliant cresyl blue. Siderocytes are seen with Prussian blue. (On Wright stain, sideotic granules are referred to as Pappenheiner bodies).


Match the following abnormalities with an associated condition.

Abnormality and Conditions:

Basophilic stippling = Lead poisoning
Howell-Jolly bodies = Post-splenectomy
Heinz bodies = G-6-PD deficiency
Pappenheimer bodies = Sideroblastic anemia
Rouleaux = Multiple myeloma
Schistocytes = Disseminated intravascular coagulation, Spherocytes = Burns


What is the clinical significance of hypochromic microcytic RBC?

They are seen in iron deficiency anemia and thalassemia and sometimes with anemia of chronic disease.


Why is it important to report even a few oval macrocytes?

Because of their association with megaloblastic anemia.


Describe rouleaux.

RBCs arranged like stacks of coins. They are the result of a serum protein abnormality, either increased globulin or fibrinogen. They are often seen in patients with multiple myeloma. Rouleaux my be erroneously reported if one examines the thick part of the smear where the RBCs are overlapping. Artifactual rouleaux may also result from delay in spreading the drop of blood while making the smear.


In what ways will the RBC morphology of a newborn differ from that of an adult?

The RBCs of newborns are normally macrocytic and polychromatophilic. Newborns may have a few nucleated RBCs (1-5/100 WBCs) which are usually gone by 3-5 days of age. Reference ranges for WBCs, RBCs, hematocrit, hemoglobin, and reticulocytes are higher for newborns than for adults.


If a patient has an MCV of 110 fL and marked polychromasia, what other hematology test would be abnormal?

The reticulocyte count would be increased.


What effect does chronic alcoholism have on RBC morphology?

The MCV is usually elevated, target cells are common, and stomatocytes may be seen.


What changes occur in a blood specimen stored at room temperature?

The RBCs swell, increasing the MCV and hematocrit and decreasing the MCHC and erythrocyte sedimentation rate. The platelets and WBCs decrease and the osmotic fragility increases.


What are the time limits for performing tests on EDTA blood?

CBCs and reticulocyte counts can be performed on blood stored at room temperature for up to four hours, but blood smears should be prepared as soon after collection as possible. Erythrocyte sedimentation rates should be set up within two hours of collection if stored at room temperature or within six hours if refrigerated. The refrigerated specimen should be brought to room temperature prior to testing.


How should EDTA specimens be mixed prior to testing?

By gentle inversion at least 60 times or by mixing on a mechanical rotator for two minutes. Shaking or vortexing lyses the RBCs. Some mechanical rockers do not adequately mix blood that has settled.


What is a supravital stain?

A stain that stains unfixed cells in the living state. Examples are new methylene blue, brilliant cresyl blue, and crystal violet.


What stain is used for reticulocytes?

New methylene blue or brilliant cresyl blue.


Where are reticulocytes found after centrifugation of whole blood?

Just below the buffy coat. They are less dense than mature RBCs.


How is the reticulocyte count determined by the manual method?

Following staining with new methylene blue, 1000 RBCs are examined and the number containing reticulum noted. (Cells must contain two or more blue-staining inclusions to be counted as reticulocytes.) The percent is determined by dividing the number of retics over 1000 RBCs by 10 (moving the decimal point one place to the left). For example, if 100 reticulocytes are counted, the reticulocyte count is 10%. Alternately, a Miller disk can be used to facilitate counting. A miller disk is a calibrated disk that is placed in the microscope ocular. RBCs are counted in the small B square and reticulocytes in the large A square which is 9 times the area of the B square. A minimum of 300 RBCs are counted, providing an estimate of the reticulocyes per 2700 RBCs. Reitculocyte % = (reticulocytes in square A x 100) / (RBCs in square B x 9). Manual reticulocyte counts are imprecise, with coefficients of variation as high as 50%. Automated reticulocyte counts are now available on some hematology analyzers.


What other RBC inclusions are seen on a reticulocyte smear?

Howell Jolly bodies, Heinz bodies, and Pappenheimer bodies are all stained by supravital stains.


What is the reference range for reticulocytes in an adult when performed by the manual method?

0.5-2%. The reference range for newborns is 2-6%. By 1-2 weeks of age, normal values are the same as for adults.


How do reticulocytes stain on a Wright stain?

As polychromatophilic RBCs.


What is the significance of a high reticulocyte count?

Increased erythropoiesis. The reticulocyte count is always elevated in hemolytic states in which there is a normal compensatory bone marrow response.


What is the corrected reticulocyte count?

A calculated correction for anemia. The corrected reticulocyte count = reticulocyte % x (patient's hematocrit (%) / normal hematocrit based on age and sex).


A 45 year old male with a hematocrit of 36% has a reticulocyte count of 5%. What is the corrected reticulocyte count?

Corrected reticulocyte count = 5% x (36/45) = 4%.


Why is the absolute reticulocyte count (ARC) becoming the preferred method of reporting reticulocytes?

It accounts for variation due to the number of RBCs. Reticulsytes x 10^9/L = [reticulocyte % x RBC count (10^12/L) x 1000] / 100. The referenge range is 25-75 x 10^9/L.


What are shift reticulocytes?

Reticulocytes that leave the bone marrow prematurely and mature in the circulation for longer than the normal maturation time of 1 day, leading to a falsely reticulocyte count.


What is the reticulocyte production index (RPI)?

A parameter that indicates the amount of erythropoiesis occurring when stress reticulocytes are present. The RPI takes into account the fact that prematurely released reticulocytes takes two days to mature instead of one. The RPI is calculated by dividing the corrected reticulocyte count by a maturation time correction factor based on the pattern's hematocrit. The correction factor is obtained from a table. Normal bone marrow activity produces an RPI of 1. An RPI > 3 represents an adequate response to anemia by the bone marrow. An RPI < 2 is considered an inadequate response to anemia by the bone marrow.


A patient with a hematocrit of 35% has a corrected reticulocyte count of 5%. The maturation time correction factor for a hematocrit of 35% is 1.5. What is the patient's RPI?

RPI = 5/1.5 = 3.3%


What is the most rapid, accurate, and precise method for reticulocyte counts?

Flow cytometry. Reticulocytes are counted on the basis of optical scatter or fluorescence after treatment with fluorescent dyes or nucleic acid stains. As many as 32,000 RBCs are counted, resulting in greatly improved precision. Automated retic counts eliminate problems inherent in manual reticulocyte count, such as stain variability, slide distribution errors, statistical sampling errors, and interobserver variability. Values are reported in absolute as well as relative terms. Reference ranges by flow cytometry are slightly higher than by the manual method. As more laboratories report automated retics, the corrected reticulocyte count and reticulocyte production index will be used less frequently.


Define PCV.

Packed cell volume, another name for hematocrit. It is the ratio of the volume of RBCs to the volume of whole blood.


How do the spin hematocrit and the hematocrit derived from an automated cell counter differ?

The automated hematocrit is a calculated value from RBC and MCV, therefore it may be somewhat lower than a spun hematocrit in which there is always some trapping of plasma within the RBC column. The difference is usually negligible unless there is a great deal of poikilocytosis and anisocytosis.


What is a normal hematocrit for a male?

42-52% (0.42-0.52 L/L). L/L is the SI unit.


What is a normal hematocrit for a female?

37-47% (0.37-0.47 L/L). L/L is the SI unit.


What is a normal hematocrit for a newborn?

53-65% (0.53-0.65 L/L). L/L is the SI unit.


What is the usual time and speed of centrifugation for a microhematocrit?

10,000 RPM for 5 minutes. Lower speeds or times lead to inadequate packing of the RBCs and a falsely elevated hematocrit. RPM should be checked periodically with a tachometer.


How can the safety of the hematocrit procedure be improved?

By using glass capillary tubes wrapped in puncture-resistant film and which do not require manually pushing one end of the tube into putty to form a plug.


While performing a manual hematocrit, the technologist observes an increased buffy coat. What is the significance of this observation?

The patient has an elevated WBC. It is important that the buffy coat not be included in the measurement of the RBC column or the hematocrit will be falsely elevated.


While reading a manual hematocrit a technician observes that the plasma is pink. How would this affect the hematocrit?

Hemolysis of the RBCs decreases the hematocrit . Blood should be redrawn and retested.


A phlebotomist had a difficult time obtaining blood from a patient and was only able to fill the EDTA tube one-fourth full. How would this affect the hematocrit?

Although the automated hematocrit would probably not be affected, a microhematocrit would be decreased because the RBCs would be crenated by the excess anticoagulant. Over-anticoagulation also decreases ESR, breaks up platelets, increases the platelet count, and causes degenerative changes in the WBCs.


A new phlebotomist had difficulty finding a vein in a patient and the tourniquet was left on for three minutes before the blood was obtained. How would this affect the hematocrit?

It would be elevated due to hemoconcentration.


Which of the RBC indices is considered most clinically useful?

Mean corpuscular volume (MCV). It indicates the average size of the RBCs and is useful in the classification of anemias. The mean corpuscular hemoglobin (MCH) usually parallels the MCV and doesn't provide any more clinically useful information. Mean corpuscular hemoglobin concentration (MCHC) is more useful as an indicator of an interfering factor in the specimen or a problem with the instrument than it is clinically. With microcytic anemia, low MCHC is not seen until the anemia is severe. The only clinical significance is an increased MCHC is hereditary spherocytosis and then the MCHC rarely exceeds 38%.


What is the formula for calculating MCV?

MCV = Hct (%) x 10 / RBC (10^12/L)


What is the formula for calculating MCH?

MCH = Hgb (g/dL) x 10 / RBC (10^12/L)


What is the formula for calculating MCHC?

MCHC = Hgb (g/dL) x 100 / Hct (%)


Calculate the MCV, MCH, and MCHC for the following specimen. How should the RBCs appear on the blood smear?
RBC 3.0 x 10^12/L
HGB 6 gm/dL
HCT 20%

MCV= (20% x 10)/3.0 = 67 fl
MCH= (6 x 10)/3.0 = 20 pg
MCHC= (6 x 100)/20% = 30 g/dL
The RBC would appear microcytic and slightly hypochromic.


What is the adult reference range for MCV?

80-96 fL.


What is the adult reference range for MCH?

27-32 pg.


What is the adult reference range for MCHC?



A patient's MCHC is reported as 37%. What abnormality should be looked for on the peripheral blood smear?

Spherocytes. Since spherocytes are the only cause of an increased MCHC, if they are not present, one or more of the RBC parameters may be incorrect. The same would be true if hypochromic RBCs were not observed on a smear of blood with a very low MCHC. Both situations require investigation.


What abnormality would be expected on the blood film when the MCV is high?



Explain how a blood sample can have a normal MCV when marked anisocytosis is observed on the blood smear.

Since MCV is an average it can be normal when there is a combination of macrocytes and microcytes. The RDW would be increased.


What abnormality would be expected on the blood film when the MCHC is low?

Hypochromic RBCs.


What are the "rules of three"?

A quality control check of the RBC parameters of the CBC. RBC x 3 = HGB +/- 0.5 and HGB x 3 = HCT +/- 2. These rules apply only to normocytic, normochromic RBCs. When values obtained do not follow the rules, one should check for abnormal RBC morphology on the blood smear, a specimen problem (ex: lipemia, cold agglutinins), or an instrument error.


A patient has a hemoglobin of 12.3 g/dL. What is the expected range for the patient's hematocrit?

36.9 +/- 2%.


What happens to RBCs in a hypertonic solution?

They crenate as water leaves the cells. In a hypotonic solution, water enters the cells and they lyse. When RBCs are counted on an automated cell counter, the diluent used must be isotonic (0.85%).


What is a normal RBC count for an adult male?

4.7-6.1 x 10^6 per uL (10^12/L).


What is a normal RBC count for an adult female?

4.2-5.4 x 10^6 per uL (10^12/L).


What does the erythrocyte sedimentation rate (ESR) measure?

The degree of settling of the erythrocytes in a column of blood in one hour. An increased ESR is a nonspecific indicator of inflammation. The ESR is increased with infections, rheumatoid arthritis, rheumatic fever, and myocardial infarction.


What are some technical sources of error for the ESR?

Technical errors that decrease the ESR include blood over two hours old, over-anticoagulation, refrigerated blood or low room temperature. Technical errors that increase the ESR include a tilted tube and high room temperature. Other sources of error are inaccurate timing or reading of the scale, or bubbles in the blood column.


What are some specimen factors that will affect the ESR?

The presence of many target cells or poikilocytes will decrease the ESR because rouleaux do not form as readily. The ESR is increased in multiple myeloma because the increased globulins favor rouleaux formation. Severe anemia will elevate the ESR. Some authors recommend correcting the ESR for the hematocrit.


What is a normal Westergren ESR for a man?

Under age 50: 0-10 mm/hr. Over age 50: 0-13 mm/hr.


What is a normal Westergren ESR for a woman?

Under age 50: 0-13 mm/hr. Over age 50: 0-20 mm/hr.


Discuss the principle of the automated ESR.

An optoelectronic sensor measures the change in opacity of a column of blood as the RBCs settle. The tubes are positioned at an angle to accelerate sedimentation so that the results can be obtained in 20 minutes instead of 60.


Explain the principle of the osmotic fragility test.

RBCs are diluted in 0-0.85% saline solutions and the amount of hemolysis at each concentration is determined by measuring the absorbance of the supernatant at 540 nm. The osmotic fragility is increased with hereditary spherocytosis and acquired hemolytic anemias with spherocytes. Since spherocytes are spheres rather than biconcave disks they are unable to expand. When water enters the cells, they lyse. The osmotic fragility is decreased in anemias where there are many target cells. Target cells are thin and are able to expand when water enters.


What are normal values for an osmotic fragility?

Hemolysis begins at 0.45% and ends at 0.30%.


What is the advantage of the incubated osmotic fragility?

It is more sensitive so it detects mild spherocytosis.


What anticoagulant should be used for an osmotic fragility?



Describe the hemoglobin molecule.

It is made of four polypeptide chains (globin) and four heme groups, each containing a protoporhyrin ring with iron.


Where is hemoglobin produced?

In the nucleated RBCs of the bone marrow.


What reagent is used in the cyanmethemoglobin method for hemoglobin?

Drabkin's reagent. Hemoglobin is converted to methemoglobin by potassium ferricyanide. The methemoglobin reacts with potassium cyanide to form cyanmethemoglobin. Absorbance is measured at 540 nm. The cyanmethemoglobin method is used for manual and automated determinations and measures all types of hemoglobin except sulfhemoglobin.


What is methemoglobin?

Hemoglobin in which Fe++ (ferrous) has been oxidized to Fe+++ (ferric). In order to bind oxygen, iron must be in the ferrous state (Fe++).


What forms of hemoglobin are normally found in the circulation?

Primarily oxyhemoglobin and deoxyhemoglobin and to a lesser extent, carboxyhemoglobin and methemoglobin. Sulfhemoglobin is not normal.


A patient with leukemia has a WBC of 120,000. How might this affect the hemoglobin determination?

It will be falsely elevated due to turbidity caused by the WBCs. This in turn will cause an inaccurate MCH and MCHC.


A patient with a hemoglobinopathy has lyse-resistant RBCs. How would this affect the hemoglobin and what should be done to obtain an accurate value?

Unlysed RBCs cause turbidity which falsely increases the hemoglobin. The blood should be diluted 1:2 with distilled water to lyse the RBCs and the hemoglobin value obtained should be multiplied by 2. (Some new analyzers have a resistant RBC lyse mode.)


What is a normal hemoglobin value for an adult male?

14-18 g/dL (140-180 g/L).


What is a normal hemoglobin value for an adult female?

12-16 g/dL (120-160 g/L).


What is a normal hemoglobin value for a newborn?

17-23 g/dL (170-230 g/L).


What are the normal hemoglobins?

Hemoglobin A, A2, and F.


Which globin chains are found in Hb A, Hb A2, and Hb F?

Hb A = alpha2beta2
Hb A2 = alpha2delta2
Hb F = alpha2gamma2


What are the normal values for adults and newborn for Hb A, Hb A2, and Hb F?

Hb A = Adult 97%, newborn 20%
Hb A2 = Adult 2%, newborn 2%
Hb F = Adult 1%, newborn 80%


What are the two broad categories of hemoglobin disorders?

Qualitative and quantitative. In qualitative disorders, a structurally abnormal hemoglobin such as Hb S or Hb C is produced. In quantitative disorders (thalassemia) only normal hemoglobins (A, A2, and F) are produced but in abnormal concentrations. (Some authors use the term hemoglobinopathy only in reference to the qualitative or structural disorders and the term thalassemia in reference to the quantitative disorders. Others use the term hemoglobinopathy to refer to both types.)


Name several conditions in which hemoglobin F is increased.

Hemoglobin F (fetal hemoglobin) is the predominant hemoglobin in the fetus and newborn. In adults, increased levels of hemoglobin F are seen with hemoglobinopathies, thalassemia, and hereditary persistence of fetal hemoglobin. The highest levels of hemoglobin F are seen with beta thalassemia major, where it is the predominant hemoglobin.


How are abnormal hemoglobins identified?

Historically, by hemoglobin electrophoresis; however an automated method by cation-exchange high performance liquid chromatography (HPLC) is now available and is being used for screening. If an abnormal hemoglobin is identified, hemoglobin electrophoresis is then performed.


Describe the migration of the most common hemoglobins on cellulose acetate at pH 8.6.

In order of migration toward the anode (fastest to slowest): A, F, S, A2 and C. (A2 and C have the same mobility.)


What controls are required for hemoglobin electrophoresis?

At least hemoglobins A, F, and S.


Why is citrate agar at acid pH used when abnormal hemoglobins are identified on cellulose acetate at pH 8.6?

It separates hemoglobins that migrate together on cellulose acetate. The order of migration from fastest to slowest is C, S, A and A2, and F.


Describe the molecular structure of hemoglobin S.

Valine is substituted fro glutamic acid in the 6th position of the beta chain.


Describe the molecular structure of hemoglobin C.

Lysine is substituted for glutamic acid in the 6th position of the beta chain.


What is the most common thalassemia in the U.S.?

Heterozygous beta thalassemia (beta thalassemia minor). It is usually a benign disorder but requires genetic counseling. Lab findings include mild anemia, increased RBCs, mild to moderate microcytosis and hypochromia, and normal RDW. The hallmark of this disorder is an elevated Hb A2 (3.5-8%).


Which hemoglobin is resistant to alkali denaturation and acid elution?

Hb F. The Kleihauer-Betke acid elution stain can be used to demonstrate Hb F. Cells containing Hb F resist acid elution and stain pink. Cells containing Hb A lose hemoglobin and appear as "ghost cells".


How does the fetus benefit from a high concentration of hemoglobin F?

Hemoglobin F has increased oxygen affinity so the fetus is able to extract oxygen from the maternal blood supply.


Describe the hemoglobin electrophoresis pattern and RBC morphology typically seen with sickle cell anemia.

On hemoglobin electrohphoresis there is approximately 80-95% Hb S, 1-10% Hb F, and 2-3% Hb A2. Because of the beta chain abnormality, no Hb A is produced. The blood smear may show sickle cells, anisocytosis, target cells, nucleated RBCs, spherocytes, Howell Jolly bodies, basophilic stippling, Pappenheimer bodies, and polychromasia. The RBCs are normocytic and normochromic.


Describe the hemoglobin electrophoresis pattern and RBC morphology typically seen with sickle cell trait.

The blood smear appears essentially normal except for a few target cells. Sickle cells are not seen except under extreme hypoxic conditions. Hemoglobin electrophoresis reveals 55-70% Hb A, 30-45% Hb S, and less than 2% Hb F.


What causes sickle cells to sickle?

When deoxygenated, Hb S polymerizes and the resulting intracellular crystals deform the RBC.


Name a reducing reagent that can be used to screen for sickle cells in a drop of fresh whole blood.

Sodium metabisulfite. The test is infrequently performed today. It has been replaced by the dithionite solubility test.


A dithionite solubility test is performed and the black lines cannot be seen through the tube due to turbidity. What should be done next?

Cellulose acetate electrophoresis. The dithionite solubility test is a screening test and is not specific for hemoglobin S. There are other rare sickling hemoglobins that give positive results. The solubility test does not differentiate SS from AS.


How is sickle cell anemia differentiated from sickle cell trait?

By hemoglobin electrophoresis.


Why can screening tests not detect sickle cell anemia in newborns?

The predominant hemoglobin in neonates, even those with sickle cell anemia, is Hb F. The amount of Hb S present newborns with sickle cell anemia is below the sensitivity of the screening tests.


Describe the electrophoretic pattern and RBC morphology typically seen with hemoglobin C disease.

Hemoglobin electrophoresis shows greater than 90% Hb C and 1-7% Hb F. Because of the beta chain abnormality, no hemoglobin A is produced. The blood smear shows numerous target cells and a few spherocytes. Hb C crystals may be observed. The RBCs are normocytic and normochromic.


Describe the electrophoretic pattern and RBC morphology typically seen with hemoglobin C trait.

50-60% Hb A, 40-50% Hb C, and less than 2% Hb F. The blood smear shows many target cells and slightly hypochromic RBCs.


Describe the electrophoretic pattern and RBC morphology typically seen with SC disease.

Electrophoresis shows approximately equal amounts of Hb S and C, with normal or slightly increased Hb F. Target cells, pocketbook cDescells, and occasional spherocytes and Hb SC crystals will be seen on the blood smear.


Describe the electrophoretic pattern and RBC morphology typically seen with beta thalassemia major.

Beta thalassemia major is a microcytic hypochromic anemia characterized by target cells, nucleated RBCs, basophilic stippling, and marked anisocytosis and poikilocytosis. There is greater than 94% Hb F and 1-6% Hb A2. No Hb A is produced.


Describe the electrophoretic pattern and RBC morphology typically seen with beta thalassemia minor.

On hemoglobin electrophoresis, there is greater than 90% Hb A, 3.5-8% Hb A2, and 1-2% Hb F. The blood smear shows hypochromic microcytic RBCs, poikilocytosis, target cells, and basophilic stippling.


Name a method other than electrophoresis that is used to quantitate Hb A2.

Chromatography--either anion exchange column chromatography or high-performing liquid chromatography.


Which anemias are microcytic and hypochromic.

Iron deficiency, beta thalassemia minor, and some anemias of chronic disease. It should be noted that in iron deficiency, anemia may be documented before microcytosis or hypochromia.


What laboratory tests help differentiate iron deficiency anemia from beta thalassemia minor?

Serum iron, total iron binding capacity (TIBC) or transferrin, % saturation, serum ferritin, RDW, RBC, and hemoglobin A2. In iron deficiency anemia the serum iron is decreased, the TIBC and transferrin are increased, and the % saturation and serum ferritin are decreased. In beta thalassemia minor these tests are normal. The RDW is increased in iron deficiency anemia and is normal in beta thalassemia minor. Hemoglobin A2 is increased in beta thalassemia minor and is normal in iron deficiency anemia. The red count is usually decreased in iron deficiency anemia and increased in beta thalassemia minor.


Why do transferrin levels increase in iron deficiency anemia?

The body is trying to capture as much iron as possible.


How is % saturation determined?

(Serum iron / TIBC) x 100. Transferrin is normally 33% saturated. Percent saturation is decreased in iron deficiency anemia and anemia of chronic disease and increased in sideroblastic anemia.


What is anemia of chronic disease (ACD)?

Anemia that results from inflammation, chronic infection, or malignancy. ACD is seen with TB, lung abscesses, bacterial endocaritis, rheumatoid arthritis, rheumatic fever, systemic lupus erythematosus, uremia, and chronic liver disease. One-fourth to 1/3 of patients have hypochromic microcytic RBCs.


How can iron deficiency anemia be differentiated from anemia of chronic disease?

The serum iron is decreased in both anemias, but the TIBC is increased in iron deficiency anemia and decreased in anemia of chronic disease. Also, the RDW is increased in iron deficiency anemia and normal in ACD.


Which anemia is characterized by a dimorphic population of RBCs?

Sideroblastic. Sideroblastic anemia is a disorder of iron utilization. Other abnormalities include pappenheimer bodies (siderotic granules), an increased number of ringed sideroblasts in the marrow, an increased number of ringed sideroblasts in the marrow, and increased serum ferritin and serum iron. Sideroblastic anemias may be inherited or acquired. Secondary sideroblastic anemias are the result of ingestion of alcohol, lead, and various medications. The peripheral blood smear often reveals microcytic, hypochromic RBCs and normocytic, normochromic RBCs.


What are ringed sideroblasts?

RBC precursors with visible iron surrounding the nucleus when stained with Prussian blue. They are the result of impaired heme synthesis.


Which test is a good indicator of the body's iron stores?

Serum ferritin. It is a surrogate marker for bone marrow iron and is measured by immunoassay. Concentrations decline early with iron deficiency anemia.


Name and explain 2 preanalytic factors that can affect iron studies.

Time of collection and the fasting state of the patient. Since iron shows a diurnal variation, with levels dropping throughout the day, the specimen should be collected early in the morning. The patient should be fasting to avoid false elevations from iron in food.


After treatment for iron deficiency anemia, which hematology test can be used to monitor the efficacy of the treatment?

An increase in the reticulocyte count is a sign of effective therapy. The reticulocyte count should increase within a few days, peaking in 7-12 days. Hemoglobin should increase in 2-3 weeks and reach normal levels within 2 months.


Which type of anemia develops with chronic blood loss?

Iron deficiency anemia. Gastrointestinal bleeding is the most common cause of iron deficiency anemia in men and in post-menopausal women.


What is the most common type of anemia in infancy, childhood, and during pregnancy?

Iron deficiency anemia.


Describe the blood picture in pernicious anemia (PA),

The blood smear shows oval macrocytes, hypersegmented neutrophils, and anisocytosis. There is leukopenia and thrombocytopenia. Pernicious anemia, a megaloblastic anemia, is due to a vitamin B12 deficiency. Atrophy of the gastric mucosa leads to decreased levels of intrinsic factor which is required for absorption of vitamin B12. Achlorhydria, decreased serum vitamin B12, extremely elevated LD-1 and 2, and elevated total and indirect bilirubin are other important diagnostic findings in pernicious anemia. Bilirubin and LD are increased because the RBCs die during division in the bone marrow, For this reason, PA can be considered a hemolytic anemia.


What chemistry tests should be ordered when hypersegmented polys and macrocytes are observed on a patient's peripheral smear?

These are signs of a megaloblastic anemia so serum B12 and folate should be ordered.


What finding is essentially diagnostic for megaloblastic anemia?

Hypersegmentation. It appears early in the disease and persists throughout treatment. Five or more 5-lobed neutrophils per 100 WBCs or any 6-lobed should be reported.


A 10-year-old patient presents with anemia, jaundice, and splenomegaly. The MCHC is slighting increased and numerous spherocytes are observed on the blood smear. What test should be performed?

An osmotic fragility. The findings are suggestive of hereditary spherocytosis.


What is the only disease in which the MCHC is elevated above the reference range?

Hereditary spherocytosis and then it is rarely above 38%.


Is the the reticulocyte count increased, decreased, or normal in hemolytic anemia?

It is increased. The bone marrow attempts to compensate for the blood loss by releasing immature RBCs.


What are the effects of G-6-PD deficiency?

It may result in a hemolytic anemia following ingestion of certain foods and drugs. Heinz bodies (denatured hemoglobin) may be formed in the RBCs.


What is paroxysmal nocturnal hemoglobinuria (PNH) and how is it diagnosed?

It is a rare chronic acquired hemolytic anemia in which the RBCs are abnormally susceptible to lysis by complement. Traditional tests for diagnosis are the sugar water screening test (sucrose hemolysis) and Ham's acidified serum test. Flow cytometry may now be used to measure CD59 on the RBCs. Hemosiderinuria is almost always seen and is of great diagnostic importance.


What is paroxysmal cold hemoglobinuria (PCH)?

It is the rarest form of autoimmune hemolytic anemia. It is caused by the Donath-Landsteiner antibody (anti-P) which binds to the RBCs during exposure to cold and causes them to lyse when they are returned to 37C. PCH is often secondary to viral diseases, especially in children.


What are some signs of hemolytic anemia?

Decreased RBCs, decreased hematocrit and hemoglobin, increased reticulocytes, polychromasia, hemoglobinemia, increased indirect bilirubin, decreased haptoglobin, increased LD, hemoglobinuria, and increased urine urobilinogen. Hemolytic anemia is a normocytic normochromic anemia.


What is hemolytic uremic syndrome (HUS)?

Acute intravascular hemolysis and renal failure that is associated with E. coli O157:H7 infection in infants and young children. Hemoglobin is severely decreased and the blood film shows schistocytes and burr cells.


Describe the blood picture in aplastic anemia.

There is a reduction in all of the cellular elements (pancytopenia). Aplastic anemia is caused by drugs, chemicals, radiation, infection, and immune disorders.


Is the reticulocyte count increased, decreased, or normal in aplastic anemia?

It is decreased.


What is Fanconi's anemia?

A congenital form of aplastic anemia. Treatment includes bone marrow transplantation.


Describe the blood picture in acute blood loss.

The white count increases within several houes as WBCs are released from the marginating and bone marrow pools. The reticulocyte count increases and nucleated RBCs may appear, depedning on the severity of the blood loss. The hemtocrit and hemoglobin are not decreased immediately following acute blood loss because RBCs and plasma are lost proportionately. Over the next several days as fluid moves into the blood vessels from the extravascular compartments the full extent of the blood loss becomes apparent.


Describe the anemia if the MCV is 85 and the MCHC is 33.

Normocytic and normochromic. Anemias of this type include hemolytic anemia, aplastic anemia, and the hemoglobinopathies.


What is polycythemia vera?

A chronic myeloproliferative disorder characterized by an increase in RBCs, WBCs, and platelets.


What is relative polycythemia?

A condition in which the hematocrit is increased due to a decrease in plasma volume. There is no increased production of RBCs.